TY - JOUR

T1 - Common kinetic mechanism of abasic site recognition by structurally different apurinic/apyrimidinic endonucleases

AU - Kuznetsova, Alexandra A.

AU - Senchurova, Svetlana I.

AU - Ishchenko, Alexander A.

AU - Saparbaev, Murat

AU - Fedorova, Olga S.

AU - Kuznetsov, Nikita A.

N1 - Funding Information: Funding: This work was supported partially by a Russian-Government–funded project (No. AAAA-A17-117020210022-4), by Electricité de France (RB 2021-05, to M.S.) and by French National Research Agency (ANR-18-CE44-0008, to A.A.I.). The part of this work involving Trp detection combined with stopped-flow kinetics was specifically funded by Russian Science Foundation grant No. 19-74-10034. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2021/8/2

Y1 - 2021/8/2

N2 - Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.

AB - Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.

KW - Abasic site

KW - Apurinic/apyrimidinic endonuclease

KW - Conformational dynamics

KW - Damaged DNA

KW - DNA repair

KW - Stopped-flow enzyme kinetics

KW - Catalytic Domain

KW - Escherichia coli

KW - Humans

KW - Substrate Specificity

KW - Molecular Dynamics Simulation

KW - Nucleotides/chemistry

KW - DNA Repair

KW - Protein Conformation

KW - DNA Damage

KW - Kinetics

KW - Nucleic Acid Conformation

KW - Binding Sites

KW - DNA Cleavage

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry

UR - http://www.scopus.com/inward/record.url?scp=85113167352&partnerID=8YFLogxK

U2 - 10.3390/ijms22168874

DO - 10.3390/ijms22168874

M3 - Article

C2 - 34445579

AN - SCOPUS:85113167352

VL - 22

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 16

M1 - 8874

ER -